Specific features of the external morphology of starry flounder Platichthys stellatus at atavism in the body color

An analysis of the morphology of starry flounder Platichthys stellatus, completely pigmented from both sides, was performed. It considerably differs from common individuals (pigmented from only one side) by several characters. The right eye in the atavistic specimen was located on the upper part of the head, and there was a deep notch between the eye and the dorsal fin. The differences between body proportions and sizes of fins in bilaterally pigmented and common flounder specimens were also detected. Based on an analysis of the data obtained, opinions concerning the causes of these morphological deviations in bilaterally pigmented flounder individuals are provided.     

Immunological detection of winter flounder (Pseudopleuronectes americanus) eggs and juveniles in the stomach contents of crustacean predators

Predation on the early life history of fish is an important factor regulating year-class strength. Verifying predation events, however, is difficult when analyses rely on visually identifying the remnants of partially digested fish in the stomachs of suspected predators. The objective of this study was to assess the utility of using immunological assays to detect the presence of winter flounder eggs and juveniles (Pseudopleuronectes americanus) in the gut contents of sand shrimp (Crangon septemspinosa) and green crab (Carcinus maenas). After defining assay capabilities, the stomach contents of field-collected shrimp and crabs were examined to determine if these predator-prey relationships occur under natural conditions. Winter flounder-specific antisera developed and used in this study successfully identified homologous antigens (egg or juvenile flounder extracts) without appreciably cross-reacting with antigenic material from predators or nontarget prey. Moreover, antisera detected flounder eggs 10.8-16.4 h after initial feeding by various sized shrimp, and identified juvenile flounder 9.4 and 7.8 h after initial ingestion by shrimp and crabs, respectively. Immuonological dietary analysis of decapod crustaceans collected from Niantic River, Connecticut, revealed that C. septemspinosa and C. maenas are potentially important predators on the early life stages of winter flounder. The temporal trends and magnitude of flounder predator-induced mortality was affected primarily by the spatial and temporal overlap between predator and prey (egg mortality), and the size-dependent relationships underlying crustacean and flatfish predator-prey interactions (juvenile mortality).     

Early life history stages of peacock flounder Bothus lunatus (Bothidae) from the western and central tropical Atlantic

Previously unknown larvae and poorly studied metamorphic and postmetamorphic stages of development of peacock flounder Bothus lunatus (Bothidae) with a length of 5.4 to 39.5 mm from the western and central tropical waters of the Atlantic Ocean are described, and their identification is substantiated. Small-and medium-sized larvae of this species (with a length of nearly 10 mm) at stages of pre-and postflexion, like all larvae of the genus Bothus at the corresponding stages of development, have a strongly flattened from the sides, moderately deep, and almost pigment-free transparent body; bear a long anterior ray in the dorsal fin; and have single melanophores in the end part of the body. They differ from larvae of other Atlantic species of the genus known at early stages of development in the shape of the anterior head profile, namely in the presence of a deep notch in front of the eyes. With growth, their body assumes a rounded shape. During metamorphosis of larval B. lunatus, the right eye shifts to the left side of the head through a slit formed during the separation of the origin of dorsal fin base from the cranium. This specific feature distinguishes them from larvae of the remaining species of the genus in which, during metamorphosis, the right eye passes to the left side through the hole in the head.     

Morphology of unfertilized mature and fertilized developing marine pelagic eggs in four types of multiple spawning flounders

Starry flounder Platichthys stellatus, spotted halibut Verasper variegates, turbot Scophthalmus maximus, and Japanese flounder Paralichthys olivaceus are four commercially cultivated multiple spawning flounders that spawn pelagic eggs. Through appropriate light and scanning electron microscope processing, the shape and surface structures (such as micropyle, pores, pore density, and paten) of unfertilized mature and fertilized developing eggs of the four species were observed and measured. First, individual or intraspecific comparisons of the surface structures of eggs at different developmental stages were made. Second, interspecific differences among the four species at the same developmental stage of unfertilized mature eggs were statistically computed and analyzed through one-way analysis of variance and hierarchical cluster analysis. Eggs of the same species collected at different stages of development tend to be different in morphology. Smoothing of the convoluted egg envelope surface and closure of the micropyle to serve as a final step of the polyspermy-preventing reaction are common after fertilization. Based on detailed morphology of micropyle of just-mature fertilizable eggs, turbot, starry flounder, and Japanese flounder each have a micropyle with a long canal but no distinct micropylar vestibule, type III of Riehl and Götting (Arch Hydrobiol 74:393–402, 1974). In contrast, spotted halibut has a micropyle with a distinct flat micropylar vestibule and a long canal, type II. Envelope surface microstructures, especially those in the micropyle region, are useful characters for egg identification among the four species. Cluster analysis using selected egg characters indicated the highest similarity between turbot and Japanese flounder and that starry flounder is obviously more similar to turbot and Japanese flounder than to spotted halibut.     

Expression and promoter activity of endogenous retroviruses in the Olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>)

Olive flounder (Paralichthys olivaceus) is regarded as one of economical fish species in the world. Genome information of Olive flounder has been revealed by next generation sequencing. Endogenous retroviruses (ERVs) as a member of transposable elements have been involved in functional roles of various genes in variety organisms. In this study, we identified an Olive flounder ERV (OF-ERV5) using RepeatMasker program, and examined expression pattern of pol gene of OF-ERV5, which indicated the high expression in two kidney samples (head kidney and body kidney). In addition, the 5′LTR sequences of OF-ERV5 are cloned into pGL4.11 vectors to confirm promoter activity. Luciferase assay indicated that the OF-ERV5_LTR showed promoter activity in both HepG2 and HINAE cell lines. These data could be of great use for further study to understand biological function of transposable elements in Oliver flounder.     

Ghrelin in the summer flounder: Immunolocalization to the gastric glands and action on plasma cortisol levels

We searched for evidence of the hormone ghrelin in the stomach of a juvenile, marine teleost, the summer flounder. Using antiserum against the conserved core of the ghrelin peptide, immunoreactivity was observed in the simple, branching epithelium that comprises the gastric glands. The immunoreaction was especially strong in the glandular epithelium located deep in the tissue. Next, we assessed a possible connection between ghrelin and the hypothalamic–pituitary–interrenal axis through a series of three injection experiments in which acylated or non-acylated (des-acyl) ghrelin was injected into the peritoneum of summer flounder (Paralichthys dentatus). A significant increase in plasma cortisol relative to saline-injected controls was observed for the acylated form at a dose of 1000 ng g^? 1 of body mass in one of the experiments. In another, there was a trend for des-acyl ghrelin at 1000 ng g^? 1 of body mass to increase plasma cortisol. Taken together, this study provides evidence that gastric glands in the stomach of summer flounder are a site of ghrelin production and that peripherally administered ghrelin can stimulate the cortisol axis in a teleost.     

Smelling Danger – Alarm Cue Responses in the Polychaete Nereis (Hediste) diversicolor (Müller, 1776) to Potential Fish Predation

The harbour ragworm, Nereis (Hediste) diversicolor is a common intertidal marine polychaete that lives in burrows from which it has to partially emerge in order to forage. In doing so, it is exposed to a variety of predators. One way in which predation risk can be minimised is through chemical detection from within the relative safety of the burrows. Using CCTV and motion capture software, we show that H. diversicolor is able to detect chemical cues associated with the presence of juvenile flounder (Platichthys flesus). Number of emergences, emergence duration and distance from burrow entrance are all significantly reduced during exposure to flounder conditioned seawater and flounder mucous spiked seawater above a threshold with no evidence of behavioural habituation. Mucous from bottom-dwelling juvenile plaice (Pleuronectes platessa) and pelagic adult herring (Clupea harengus) elicit similar responses, suggesting that the behavioural reactions are species independent. The data implies that H. diversicolor must have well developed chemosensory mechanisms for predator detection and is consequently able to effectively minimize risk.     

Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1–8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species.     

Asymmetry of the interrenal gland in two flounder species in the spawning period

Significant differences in the area of cell nuclei of the left and right lobes of the interrenal glad have been revealed within the spawning period in two flounder species—the the barfin plaice Liopsetta pinnifasciata and the Korean flounder Glyptocephalus stelleri, which testifies to the different functional activities of these lobes and, accordingly, about the functional asymmetry of the interrenal glad. Individuals with a functionally more active right lobe of the gland prevail for the barfin plaice, whereas with the Korean flounder the number of individuals with a more active left lobe is equal to the number of individuals with a more active right lobe.     

Female maturity,reproductive potential,relative distribution,and growth compared between arrowtooth flounder (Atheresthes stomias) and Kamchatka flounder (A. evermanni) indicating concerns for management

Arrowtooth flounder (Atheresthes stomias) and Kamchatka flounder (A. evermanni), major piscivorous predators in the eastern Bering Sea and Aleutian Islands, are morphologically similar. Consequently the two species have been managed together as a species complex using the length‐ and age‐at‐maturity derived from Gulf of Alaska arrowtooth flounder, which had been the only available maturity estimates. However, there could be serious management consequences if the two species matured at significantly different ages and fork lengths. Therefore, this study was conducted during 2007 and 2008 to determine if there were significant differences in maturation between the two species. Significant differences in size and age of female maturation and growth were found. The age and length of 50% maturity (A_50,L_50, respectively) for arrowtooth flounder females is 7.6 years of age and 480 mm in body length. In comparison, A_50,L₅₀ of Kamchatka flounder females is 10.1 years of age and 550 mm, meaning that Kamchatka flounder has a significantly lower reproductive potential than arrowtooth flounder. The large difference in reproductive potential indicates that managing the two species together as a species complex using the reproductive characteristics of arrowtooth flounder, was not conservative for Kamchatka flounder. This study also determined that arrowtooth flounder maturation was consistent between the Gulf of Alaska and eastern Bering Sea populations.     

Process of pigment cell differentiation in skin on the left and right sides of the Japanese flounder,Paralichthys olivaceus,during metamorphosis

Two groups of larvae of the Japanese flounder,Paralichthys olivaceus, were reared in the laboratory. The survivors of the first group (normal) showed normal pigmentation, and the second group (albinic) exhibited nearly complete pseudoalbinism after metamorphosis. The process of pigment cell differentiation on the left and right sides was observed mainly by transmission electron microscope (TEM) in relation to metamorphosis. In the normal group, chromatoblasts in the left side skin differentiated successfully, but those in the right side skin showed shrinkage and collapse during metamorphosis. Mucus cells are known as typical cells of ocular side skin in flatfish. The ratio of mucus cell density (left side/right side) increased from the onset of metamorphosis. These results suggest, some components of skin changed asymmetrically in process of metamorphosis before differences in fine structures of chromatoblasts were detected between the left and right sides of the normal group. However, in the albinic group, the same process of chromatoblast collapse occurred on the left and right sides, and there was no change in the ratio of mucus cell density during metamorphosis.     

Feeding ability and survival during starvation of marine fish larvae reared in the laboratory

The larvae of Clyde and Baltic herring (Clupea harengus L.), cod (Gadus morhua L.) and flounder (Platichthys flesus L.) were reared and fed to examine the changes in feeding ability and survival during progressive starvation. The time to initial feeding for yolk-sac larvae and to the point-of-no-return (PNR, when 50% of the larvae, although still alive, are no longer strong enough to feed) for both yolk-sac and older larvae were determined. The yolk-sac larvae of Clyde and Baltic herring, cod and flounder begin to feed on days 6, 3, 5 and 6 post-hatching at rearing temperatures of 7.5, 9.2, 6.9 and 9.5°C, respectively. The time to reach the PNR for yolk-sac larvae of these species is only 3–5 days after yolk resorption. From the onset of starvation in older larvae the time to reach the PNR is 6–7 days for 36-and 60-day-old Clyde herring at 9.6 and 10.5°C and for 46-day-old Baltic herring at 13.1°C but it is 23 days for 32-day-old flounder at 12.3°C. In yolk-sac larvae the peak of feeding rate and intensity usually occurred on the day that the yolk became exhausted, or 1 day later. Older larvae could withstand longer periods without food than yolk-sac larvae, especially in flounder. While the feeding rate during starvation of older larvae slowly decreased the feeding intensity first increased significantly and then decreased. Survival of larvae remained high up to the PNR.     

Molecular cloning and characterization of Toll-like receptor 14 in Japanese flounder,Paralichthys olivaceus

Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. TLR14, which is unique to fish, has been identified in several fish species, but its function is unclear. In this study, Japanese flounder (Paralichthys olivaceus) TLR14 gene (JfTLR14) was cloned and its expression profiles were analyzed after infection with viral hemorrhagic septicemia virus, gram-positive Streptococcus iniae and gram-negative Edwardsiella tarda. The coding region of JfTLR14 cDNA was 2,607 bp, encoding 878 amino acid residues. JfTLR14 was highly expressed in head kidney of healthy flounder. In response to infection with VHSV and S. iniae, the JfTLR14 gene was up-regulated at only 1 day post-infection (dpi). However, E. tarda infection increased JfTLR14 gene expression from 1 to 6 dpi. These results imply that JfTLR14 participates more in the immune response against E. tarda infection than in the immune responses to other pathogen infections.     

Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.     

Eimeria tenella: specific reversal of t-butylaminoethanol toxicity for parasite and host by choline and dimethylaminoethanol.

t-Butylaminoethanol is an anticoccidial compound that is related structurally to the metabolically active substances, dimethylaminoethanol, and choline. Toxic effects of t-butylaminoethanol for chickens and Eimeria tenella are specifically overcome by feeding sufficient amounts of dimethylaminoethanol or choline. Dietary concentrations of the two above metabolites required to totally overcome toxic effiects of t-butylaminoethanol were determined and are expressed as the reversal ratio, inhibitor (t-butylamino-ethanol): metabolite. The inhibitor:choline ratio for total reversal of toxic effects of the inhibitor in chickens is approximately 1:10 over a concentration range of inhibitor from 0.019 to 0.05%. The inhibitor:choline ratio for reversal of antiparasitic effects is approximately 1:200 with a concentration of 0.01% inhibitor. The inhibitor:Dimethylaminoethanol ratio for reversal of toxic effects of the inhibitor in the chicken is approximately 1:7 with a concentration of 0.015% inhibitor. The inhibitor:dimethylaminoethanol ratio for reversal of antiparasitic effects is approxmately 1:20 wth a concentration of 0.01% inhibitor.     

Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus)

Background

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20–25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA.

Methodology/Principal Findings

Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis.

Conclusions/Significance

We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.